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Method of Improving Resistance to Substrate Analog of Nitric Acid in Microalga

a technology of nitric acid and substrate analog, which is applied in the field of improving the resistance of a substrate analog of nitric acid in a microalga, can solve the problems of difficult to avoid the growth of non-targeted microorganisms owing to contamination arising after the treatment, increased energy and equipment required for sterilization or germicidal treatment of culture medium and the like, and high risk of contamination

Pending Publication Date: 2021-04-08
KAO CORP
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is related to improving the resistance of microalgae to nitric acid analogues by deleting or downregulating the expression of certain genes that are involved in the process of nitric acid uptake and reduction. This may be achieved by using genetic engineering techniques that lead to a transformation of the microalgae with resistance to nitric acid analogues. The transformation can be performed by deleting or downregulating the expression of genes encoding proteins involved in nitric acid uptake and reduction. The technical effect of this invention is the creation of microalgae that can withstand higher levels of nitric acid analogues, which could be useful in various applications such as agriculture or industrial production.

Problems solved by technology

However, as scale of microorganism cultivation is expanded, the cost of energy and equipment required for the sterilization or germicidal treatment of the culture medium and the like increases.
Further, even if culture medium subjected to sterilization or germicidal treatment is used, growth of non-targeted microorganisms owing to contamination arising after the treatment is hard to avoid.
When microorganisms (typically microalgae) are cultured in an open area using an open-type open pond or the like, risk of contamination is particularly high.
However, a strain prepared by introducing a foreign gene corresponds to a gene recombinant, and use thereof is restricted under the regulatory requirements of the Cartagena Protocol, for example, owing to its risk of spreading the foreign gene across the natural environment.
Further, the microalgae attract attention as next-generation biomass resources, because the microalgae do not compete with foods.
However, nothing has been reported regarding use of endogenous gene of microalgae in the class Eustigmatophyceae to impart drug resistance.

Method used

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  • Method of Improving Resistance to Substrate Analog of Nitric Acid in Microalga
  • Method of Improving Resistance to Substrate Analog of Nitric Acid in Microalga
  • Method of Improving Resistance to Substrate Analog of Nitric Acid in Microalga

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first embodiment

[0048]In the present invention, an NRT gene on the genome of a specific microalga is deleted. Alternatively, expression of the NRT gene encoded in the genome of the specific microalga is downregulated. The NRT gene described later is deleted or expression thereof is downregulated in the specific microalga, whereby resistance to the substrate analog of nitric acid, preferably chloric acid resistance, influencing viability of the microalga, is improved. The transformant of the present invention can also be selected using improved resistance to the substrate analog of nitric acid (preferably chloric acid resistance) as an indicator.

[0049]Further, as mentioned above, a chlorite ion formed by reduction of a chlorate ion by nitrogen metabolism exhibits high toxicity to ordinary microorganisms. However, the transformant of the present invention has high resistance to chloric acid. Therefore, when the transformant of the present invention is cultured in a chloric acid-containing medium, con...

second embodiment

[0063]In the present invention, in addition to the above-mentioned NRT gene, the NR gene is also deleted or expression thereof is downregulated. These genes are deleted or expression thereof is downregulated, whereby the resistance to the substrate analog of nitric acid is further improved. Therefore, the obtained transformant can grow even under conditions containing the substrate analog of nitric acid, preferably chloric acid with a higher concentration. Accordingly, the transformant of the present invention can be selected using the resistance to the substrate analog of nitric acid (preferably chloric acid resistance) as the indicator. Here, the term “NR” herein means an enzyme which reduces nitrate ion to form nitrite ion. Moreover, the NR reduces chlorate ion as the substrate analog of nitrate ion to form chlorite ion.

[0064]As termed in the present specification “NR gene” means not only gene including DNA formed of the nucleotide sequence in the region encoding NR but also DNA ...

example 1

Providing Chioric Acid Resistance for Nannochloropsis Oculata

(1) Construction of Plasmid for Zeocin Resistance Gene Expression

[0103]A zeocin resistance gene (SEQ ID NO: 1) was artificially synthesized. Using the thus-synthesized DNA fragments as a template, and a pair of the primer Nos. 2 and 3 shown in Table 1, PCR was carried out, to amplify the zeocin resistance gene.

[0104]Further, using a genome of Nannochloropsis oculata strain NIES-2145 (obtained from National Institute for Environmental Studies (NIES)) as a template, and a pair of the primer Nos. 4 and 5, and a pair of the primer Nos. 6 and 7 shown in Table 1, respectively, PCRs were carried out to amplify the VCP1 promoter sequence (SEQ ID NO: 8) and the VCP1 terminator sequence (SEQ ID NO: 9).

[0105]Furthermore, using a plasmid vector pUC118 (manufactured by Takara Bio) as a template, and a pair of the primer Nos. 10 and 11 shown in Table 1, PCR was carried out to amplify the plasmid vector pUC118.

[0106]The thus-obtained fo...

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Abstract

A method of improving resistance to a substrate analog of nitric acid in a microalga, containingdeleting a gene encoding the following protein (A) or (B) present in the genome of the microalga, or downregulating expression of a gene encoding the following protein (A) or (B):(A) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 41; and(B) a protein consisting of an amino acid sequence having 70% or more identity with the amino acid sequence of the protein (A), and having nitrate transporter activity.

Description

TECHNICAL FIELD[0001]The present invention relates to a method of improving resistance to a substrate analog of nitric acid in a microalga, a transformant of a microalga having resistance to a substrate analog of nitric acid, and a method of preparing a transformant having resistance to a substrate analog of nitric acid.BACKGROUND ART[0002]Culture of microorganisms for producing a biofuel or other useful substance is generally performed on the assumption of culturing pure culture of target microorganisms. Accordingly, in order to prevent contamination by microorganisms other than the desired microorganisms, sterilization treatment or germicidal treatment is usually applied to the culture medium or incubator.[0003]However, as scale of microorganism cultivation is expanded, the cost of energy and equipment required for the sterilization or germicidal treatment of the culture medium and the like increases. Further, even if culture medium subjected to sterilization or germicidal treatme...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N1/12
CPCC12N1/12C07K14/195
Inventor WADA, MAYUMIOZAKI, TATSURO
Owner KAO CORP
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